Walter Berón

Coxiella burnetii Localizes in a Rab7-Labeled Compartment with Autophagic Characteristics

ABSTRACT The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

Phagocytosed Live Listeria monocytogenes Influences Rab5-regulated in Vitro Phagosome-Endosome Fusion

Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly−). Unlike the wild-type organism, LMhly− remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly− and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly− phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly− phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly−-containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly−-containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly−-containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.

Calmodulin Regulates Endosome Fusion

Calmodulin (CaM) has previously been implicated in regulated exocytosis, transcytosis, and receptor recycling. We have investigated the role of CaM in endocytic transport by examining the effects of several CaM antagonists in intact cells. We present evidence indicating that the mixing of sequentially internalized ligands is inhibited by CaM antagonists, indicating that CaM may play a general role in regulating endosomal membrane trafficking. To address the specific events that are affected by CaM we studied its role in an in vitro assay that reconstitutes fusion among endosomes. CaM antagonists inhibited endosome fusion, and the inhibition was reversed by the addition of CaM. Moreover, we found that Ca2+ stimulates fusion among endosomes and that addition of CaM stimulates fusion beyond that produced by Ca2+ alone. Our data indicate that one of the possible targets for CaM in endosome fusion is the CaM-dependent kinase II. We propose that CaM regulates endocytic transport by modulating an essential component(s) of the membrane traffic machinery.

The actin cytoskeleton participates in the early events of autophagosome formation upon starvation induced autophagy

Autophagy is a process by which cytoplasmic material is sequestered in a double-membrane vesicle destined for degradation. Nutrient deprivation stimulates the pathway and the number of autophagosomes in the cell increases in response to such stimulus. In the current report we have demonstrated that actin is necessary for starvation-mediated autophagy. When the actin cytoskeleton is depolymerized, the increase in autophagic vacuoles in response to the starvation stimulus was abolished without affecting maturation of remaining autophagosomes. In addition, actin filaments colocalized with ATG14, BECN1/Beclin1 and PtdIns3P-rich structures, and some of them have a typical omegasome shape stained with the double FYVE domain or ZFYVE1/DFCP1. In contrast, no major colocalization between actin and ULK1, ULK2, ATG5 or MAP1LC3/LC3 was observed. Taken together, our data indicate that actin has a role at very early stages of autophagosome formation linked to the PtdIns3P generation step. In addition, we have found that two members of the Rho family of proteins, RHOA and RAC1 have a regulatory function on starvation-mediated autophagy, but with opposite roles. Indeed, RHOA has an activatory role whereas Rac has an inhibitory one. We have also found that inhibition of the RHOA effector ROCK impaired the starvation-mediated autophagic response. We propose that actin participates in the initial membrane remodeling stage when cells require an enhanced rate of autophagosome formation, and this actin function would be tightly regulated by different members of the Rho family.

Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells.

We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated β-glucuronidase-conjugated paramagnetic beads (1–2 μm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPγS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.

Heterotrimeric G Proteins Interact with the Small GTPase ARF POSSIBILITIES FOR THE REGULATION OF VESICULAR TRAFFIC

Trimeric G proteins have emerged as important regulators of membrane trafficking. To explore a role for Gβγ in endosome fusion, we have taken advantage of β-adrenergic receptor kinase (βARK), an enzyme translocated to membranes by interaction with Gβγ. The COOH terminus of βARK (βARKct) has a Gβγ-binding domain which blocks some Gβγ-mediated processes. We found that βARKct and peptide G, a peptide derived from βARKct, inhibit in vitro endosome fusion. Interestingly, peptide G and ARF share sequence similarity. Peptide G and βARKct reversed ARF-mediated inhibition of endosome fusion and blocked ARF binding to membranes. Using an ARF fusion protein, we show that both Gβγ and Gαs interact with the small GTPase ARF, an interaction that is regulated by nucleotide binding. We conclude that G proteins may participate in the regulation of vesicular trafficking by directly interacting with ARF, a cytosolic factor required for transport.

Microtubules Mediate Cellular 25-Hydroxyvitamin D3 Trafficking and the Genomic Response to 1,25-Dihydroxyvitamin D3 in Normal Human Monocytes

The genomic actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the intracellular vitamin D receptor (VDR). Although immunocytochemistry has shown that disruption of microtubular assembly prevents nuclear access of the sterol-VDR complex, the role of microtubules in the response to 1,25(OH)2D3 has not been studied in viable cells. Our studies examined this interaction in normal human monocytes. Monocytes convert 25(OH)D3 to 1,25(OH)2D3 and to 24-hydroxylated metabolites more polar than 1,25(OH)2D3. Microtubule disruption totally abolished the ability of exogenous 1,25(OH)2D3 to suppress its own synthesis and to induce 24-hydroxylase mRNA and activity, without affecting either total 1,25(OH)2D3 uptake or maximal 1,25(OH)2D3-VDR binding. Thus, intact microtubules are essential for 1,25(OH)2D3-dependent modulation of gene transcription. Interestingly, microtubule disruption also decreased monocyte 1,25(OH)2D3 synthesis, not by decreasing the Vmax of monocyte mitochondrial 1α-hydroxylase but through an increase in the Km for 25(OH)D3. We examined 25(OH)D3 transport. Microtubule disruption did not affect total cellular 25(OH)D3 uptake but reduced its intracellular trafficking to the mitochondria. Thus, microtubules participate in intracellular 25(OH)D3 transport, and their integrity determines normal 1,25(OH)2D3 synthesis.

Actin dynamics and Rho GTPases regulate the size and formation of parasitophorous vacuoles containing Coxiella burnetii.

ABSTRACT Q fever is a disease caused by Coxiella burnetii. In the host cell, this pathogen generates a large parasitophorous vacuole (PV) with lysosomal characteristics. Here we show that F-actin not only is recruited to but also is involved in the formation of the typical PV. Treatment of infected cells with F-actin-depolymerizing agents alters PV development. The small PVs formed in latrunculin B-treated cells were loaded with transferrin and Lysotracker and labeled with an antibody against cathepsin D, suggesting that latrunculin B did not affect vacuole cargo and its lysosomal characteristics. Nevertheless, the vacuoles were unable to fuse with latex bead phagosomes. It is known that actin dynamics are regulated by the Rho family GTPases. To assess the role of these GTPases in PV formation, infected cells were transfected with pEGFP expressing wild-type and mutant Rac1, Cdc42, and RhoA proteins. Rac1 did not show significant PV association. In contrast, PVs were decorated by both the wild types and constitutively active mutants of Cdc42 and RhoA. This association was inhibited by treatment of infected cells with chloramphenicol, suggesting a role for bacterial protein synthesis in the recruitment of these proteins. Interestingly, a decrease in vacuole size was observed in cells expressing dominant-negative RhoA; however, these small vacuoles accumulated transferrin, Lysotracker, and DQ-BSA. In summary, these results suggest that actin, likely modulated by the GTPases RhoA and Cdc42 and by bacterial proteins, is involved in the formation of the typical PV.

Inhibition of endocytic transport by aluminum fluoride implicates GTPases as regulators of endocytosis.

It is now well established that GTP-binding proteins are important regulators of vesicular transport. Recent work has shown that multiple GTPases (both monomeric and heterotrimeric) are required for trafficking. In the present study we have used aluminum fluoride (AIF), a reagent that activates trimeric G proteins, as a tool to study the involvement of this family of GTPases in the regulation of endocytosis in intact cells. Our results indicate that AIF inhibits fusion of early endosomes with an intracellular proteolytic compartment. Using the mixing of sequentially internalized ligands as a measure of endocytosis, we found that AIF inhibited endocytic transport as assessed by both biochemical and morphological methods. Taken together these results suggest that AIF affects membrane fusion, a common step in vesicular transport. To further examine the effects of AIF we tested this compound in a cell-free assay that reconstitutes fusion among endosomes. AIF affected endosomal fusion in a different way than did GTP gamma S, an agent that activates both trimeric and small GTPases. Our results suggest that the coordinated activation of both classes of GTPases are required for efficient endocytic transport.